The ultimate means for stopping the HIV epidemic is a prophylactic vaccine that blocks virus transmission in the general population. It is now accepted that such a vaccine will have to elicit anti-HIV antibody responses as well as cellular immunity to provide protection. What is desperately needed to meet this goal is an immunogen that will elicit broadly neutralizing antibodies capable of preventing (neutralizing) infection by primary HIV strains found worldwide. Our approach toward developing such an immunogen is based on the "constrained" transition state structure of gpl20 that is induced by CD4 binding. In previous studies, we showed that crosslinked gpl20-soluble CD4 complexes elicited antibodies in macaques that neutralized a wide variety of primary isolates regardless of Clade. We also showed that these "broadly neutralizing" antibodies were isolated from immune sera by affinity chromatography with a constrained single chain complex (called SCBaL/M9) containing gpl20 linked to a CD4 mimetic miniprotein (CD4M9). Thus, single chain gpl20-CD4 mimetic complexes warrant exploration as vaccine subunit immunogens to safely elicit broadly neutralizing antibodies in humans. However, in preliminary experiments SCBaL/M9 elicited broadly neutralizing antibodies less efficiently than a single chain gpl20-CD4 complex (FLSC). Such inefficiency can be logically interpreted to mean that a variable fraction of the SCBaL/M9 immunogen loses its intrachain complex and fails to present a constrained gpl20 structure. Such instability is predicted given that the CD4M9 sequence has a binding affinity for gp 120 that is only about 1% that of CD4. In agreement, other preliminary studies show that the intramolecular interactions in SCBaL/M9 are less stable than in FLSC. Accordingly, our central hypothesis, which will be evaluated in this project, is that the immunogenicity of SCBaL/M9 will be improved by sequence modifications that produce highly stabilized intramolecular complexes. In order to explore this hypothesis, Aim 1 of this project will be to design and evaluate modified versions of SCBaL/M9 for improved intrachain binding stability. Aim 2 will be to compare the immunogenicity of modified complexes versus SCBaL/M9 in rabbits. We expect these efforts to yield new candidate immunogens that can be feasibly used to elicit broadly neutralizing antibodies in a variety of vaccine contexts. [unreadable] [unreadable]